1. Field
The present invention relates to a spectroscopic measurement method for determining analytical high resolution NMR spectra suited for structure determination and/or structure control of chemical compounds. A further aspect of the invention concerns a sample container adapted for being placed in the sample site of an NMR measurement apparatus for use in the mentioned method.
2. Description
NMR spectroscopy techniques are a part of most molecular structure determination and verification processes in all fields of chemical and bio-structural research. The emerging field of combinatorial chemistry, with its need to characterize large compound libraries used for screening in bio-assays, and the demand for quality control of compound depositories in the pharmaceutical industry, demonstrate that sample throughput achievable with the typical serial setups used in modern NMR laboratories is too low. This low throughput now limits the application of these techniques in the above mentioned contexts. The following summarizes the two serial procedures currently in use in analytical NMR laboratories:
Procedure using an Automatic Sample Changer:
(I) Solvation of substances to be investigated in 600 xcexcl of NMR suitable (deuterated) solvent to a concentration of typically 10 mM.
(II) Transfer of each sample to a NMR sample tube
(III) Positioning of up to 120 prepared samples in the carrousel of the automated sample changer.
(IV) Serial transfer of one sample after the other to the high field magnet and setting of experimental parameters (locking, shimming).
(V) Acquisition (and storage) of NMR data.
(VI) Back transfer of the samples to the sample changer.
(VII) Emptying of the sample changer.
(VIII) Disposal of samples and rinsing of (expensive) sample tubes.
For tasks (I)-(III) and (VII)-(VIII) manual and automated versions are found in NMR laboratories. Only tasks (IV)-(V) are always fully automated. The whole procedure typically takes about 10 min. per sample. Out of these tasks, about 6 mins. [tasks (IV)-(VII)] involve costly xe2x80x9cNMR-timexe2x80x9d, whereby the actual detection of the signal takes only 1 min. Sample preparation and disposal can be done xe2x80x9coff-linexe2x80x9d and does not incur NMR-time.
To increase throughput, flow-through probe-heads have been developed and installed. In this setup a sample preparation robot is connected via a transfer capillary directly to the NMR detection cell. This procedure referred to as flow-injection NMR can be summarized as follows:
Flow Through Setup [Spraul, M., et al., Anal. Commun., 34: 339-341 (1997)]:
(I) Solvation of substances to be investigated in 300 xcexcl of NMR suitable (deuterated) solvent to a concentration of typically 10 mM in 96 well plates.
(II) Aspiration of each sample from a well to the capillary line and transfer to the NMR detection cell.
(III) Preparation of experimental parameters (locking, shimming).
(IV) Acquisition (and storage) of NMR data.
(V) Disposal of samples in waste and rinsing of the syringe and capillary system to prevent carry over of substance to the next NMR measurement.
Again, task (I) can be done manual or automated. The remaining tasks are fully roboterized. This procedure typically takes about 8 mins. per sample. Out of these 8 mins, about 5 mins. [tasks (II)-(V)] involve costly xe2x80x9cNMR-timexe2x80x9d. Only the sample preparation step can be done xe2x80x9coff-linexe2x80x9d. Again the detection of the signal takes only 1 min.
From the above summary, it is obvious that throughput rate is not limited by the NMR experiment itself but by the time necessary to position and remove the NMR sample in/from the detection volume.
An improvement in this respect is described in the literature where four detection volumes are mounted into a home-built flow-through probehead. The application of an inhomogeneous magnetic field along the z-axis together with a tailored data deconvolution method is used to measure four samples in parallel [MacNamara, E., et al., Anal. Chim. Acta, 397: 9-16 (1999)].
This method, however, is restricted to measurements of only a few samples in parallel as it necessitates a coil around each sample tube. For the same reason, a particularly adapted NMR probehead is needed.
Another problem of the current NMR methodology arises from the fact that a small portion of the available amount of material has to be consumed by the preparation of a NMR dedicated sample. For standard chemical analyses, this is typically not of much concern because for each structure verification only a single sample has to be prepared. But for the stability characterization of substances in large compound libraries, the problem is very serious. If each compound of a library is monitored once per year by NMR for structural integrity and each detection takes about 5% of the sample, then the complete library will be consumed within 20 years, a fact not often appreciated by the scientist responsible for the creation of the library.
According to U.S. Pat. No. 5,221,518, the contents of which are hereby incorporated by reference, a space-resolved NMR spectrum of a multiplicity of DNA samples can be obtained by labeling the nucleotides. However, this is only a subordinate aspect of the described invention. The obtained NMR signals can be used to detect the presence or absence of the respective labels, yet do not allow the structure to be determined, i.e. an analytical NMR signal cannot be obtained. The value of 80,000 samples, which the method should be able to measure simultaneously, is more theoretical than practical in nature. U.S. Pat. No. 4,520,316, the contents of which are hereby incorporated by reference, discloses a method for determining NMR spectra of, for example, 7 samples simultaneously. The method comprises measuring a number of NMR spectra with different field gradients applied. For calculating the NMR spectrum at a certain site, the spectra are shifted in frequency according to the respective magnetic field at the site. By comparing and adding, if predefined correlation criteria are met, the NMR spectrum of the site can be determined. Hence, this method avoids methods like Fourier transformation.
The main disadvantage of this method is that it does not provide increased throughput with regards to measuring the samples individually. In the example, it took 73 minutes to perform the needed measurements of the seven samples. Moreover, the method disclosed in this document is intended for use with tomographic scanners, i.e. for examining bodies of significant volume, and not for analytical NMR applications. The obtained NMR spectra are, therefore, not high resolution spectra, though clearly separate signals are resolved.
According to U.S. Pat. No. 4,703,270, the contents of which are hereby incorporated by reference, it is possible to determine zero-quantum-spectra simultaneously in sample sets. Measuring this kind of NMR spectra is meant to be insensible to inhomogenities of the magnetic field. However, this spectrum is determined indirectly, produces a much smaller signal, and an important kind of signal, namely those of singulett states (e.g. methyl-H), cannot be observed at all. The decreased signal amplitude requires increased measuring times, and the lack of singulett signals renders it impossible to derive the structure from the substance measured. In the example, the samples are contained in capillaries of 6 mm diameter, that is, of the usual NMR capillary size, and are arranged at a significant distance from each other. Thereby, the signal amplitudes are increased and the spatial resolution facilitated.
Accordingly, there is a need for a method that allows rapid simultaneous determination for a multiplicity of samples of the analytical spectra for structure control or structure determination. Similarly, there has been a long felt need for an NMR technique that reduces the sample volume required.
The subject invention provides a method for determining analytical high resolution NMR spectra for each of at least two chemical compounds that are contained within individual samples. These NMR spectra are suitable for structure determination or structure control of each of the at least two chemical compounds. The subject method comprises: (a) placing at least two samples, each containing one of the at least two chemical compounds, within the detection volume of a detection coil in an NMR measuring apparatus; (b) applying at least one spatially inhomogeneous magnetic field that penetrates the at least two samples within the detection volume of the detection coil in the NMR measuring apparatus, the at least one spatially inhomogeneous magnetic field having certain predetermined characteristics; (c) measuring the NMR after applying the at least one spatially inhomogeneous magnetic field, the characteristics of the inhomogeneous magnetic field, including the degree of spatial inhomogeneity, being set on at least two values to be measured; and (d) resolving the NMR signals picked-up by the detection coil during the measuring of the NMR using a spatially-resolving computational method, thereby determining the analytical high resolution NMR spectra for each of at least two chemical compounds.
Typically, the spatially resolving computational method comprises at least one Fourier transformation with respect to the variation of the inhomogeneity of the magnetic field. The Fourier transformation with respect to the variation of the inhomogeneity of the magnetic field is generally one Fourier transformation per space coordinate. In the subject invention one detection coil can encompass all of the samples penetrated by the magnetic field and all of the samples can be encompassed in the detection volume of the NMR measuring apparatus.
Preferably, at least two spatially inhomogeneous magnetic fields are applied, and the spatially-resolving computational method yields NMR signals with respect to the coordinates of the at least two spatially inhomogeneous magnetic fields in order to determine the individual NMR signals of the samples in a two-dimensional arrangement. More preferably, at least three spatially inhomogeneous magnetic fields are applied, and the spatially-resolving computational method yields NMR signals with respect to the coordinates of the at least three spatially inhomogeneous magnetic fields in order to determine the individual NMR signals of samples in a three-dimensional arrangement.
The subject invention further provides a sample container adapted for being placed in the detection volume of an NMR measurement apparatus. This sample container comprises at least two compartments that are configured and dimensioned to each contain a sample. The at least two compartments are configured, dimensioned and arranged so that NMR signals emitted by samples contained within the at least two compartments can be resolved by applying a spatially resolving computational method to NMR signals measured by a NMR measurement apparatus into which the sample container has been introduced.
Favorably, the sample container comprises at least seven compartments, for example, at least nine compartments, or at least nineteen compartments. The compartments can be NMR-suited capillary tubes that have an open inlet end and an open outlet end, the open inlet end and the open outlet end being configured and dimensioned so that they are connectable to a sample supply and a sample disposal, respectively. Alternatively, the compartments can be NMR-suited capillary tubes that are closed at one end and open at the other end, the capillary tubes being arranged as a bundle of generally circular cross-section so as to imitate a single sample tube.
Preferably, the capillary tubes have a volume of about 30 xcexcl or less, and have a cross-sectional area of at most 1 mm2.
The sample container can also comprises an adapter that is configured and dimensioned such that the sample container can be held in the detection volume of an analytical NMR measurement device in replacement of a conventional NMR tube. Favorably, the sample container comprises a three-dimensional arrangement of sample compartments. In one embodiment, a hull encompasses the sample compartments. Such a hull can be configured and dimensioned so as to produce interstices between the compartments and between the hull and the compartments, and the interstices can be fillable with a liquid. Preferably, the interstices are closed and filled with a liquid of adapted magnetic susceptibility in order to minimize disturbing the magnetic field in passing the walls of the compartments and the hull.